Hepatitis C virus (HCV) infection is an important clinical problem worldwide. In the United States alone, an estimated four million individuals are chronically infected with HCV. HCV, the major etiologic agent of non-A, non-B hepatitis, is transmitted primarily by transfusion of infected blood and blood products (Cuthbert et al., 1994, Clin. Microbiol Rev. 7:505-532). Prior to the introduction of anti-HCV screening in mid-1990, HCV accounted for 80-90% of posttransfusion hepatitis cases in the United States. A high rate of HCV infection is also seen in individuals with bleeding disorders or chronic renal failure, groups that have frequent exposure to blood and blood products.
Acute infection with HCV results in persistent viral replication and progression to chronic hepatitis in approximately 90% of cases. For many patients, chronic HCV infection results in progressive liver damage and the development of cirrhosis. In patients with an aggressive infection, cirrhosis can develop in as little as two years, although a time span of 10-20 years is more typical. In 30-50% of chronic HCV patients, liver damage may progress to the development of hepatocellular carcinoma. In general, hepatocellular carcinoma is a late occurrence and may take greater than 30 years to develop (Bisceglie et al., 1995, Semin. Liver Dis. 15:64-69). The relative contribution of viral or host factors in determining disease progression is not clear.
HCV is an enveloped virus containing a positive-sense single-stranded RNA genome of approximately 9.5 kb. On the basis of its genome organization and virion properties, HCV has been classified as a separate genus in the family Flaviviridae, a family that also includes pestiviruses and flaviviruses (Alter, 1995, Semin. Liver Dis. 15:5-14). The viral genome consists of a lengthy 5′ untranslated region (UTR), a long open reading frame encoding a polyprotein precursor of approximately 3011 amino acids, and a short 3′ UTR. The polyprotein precursor is cleaved by both host and viral proteases to yield mature viral structural and nonstructural proteins. HCV encodes two proteinases, a zinc-dependent metalloproteinase, encoded by the NS2-NS3 region, and a serine proteinase encoded in the NS3/NS4 region. These proteinases are required for cleavage of specific regions of the precursor polyprotein into mature peptides. The carboxyl half of nonstructural protein 5, NS5B, contains the RNA-dependent RNA polymerase. The function of the remaining nonstructural proteins, NS4B, and that of NS5A (the amino- terminal half of nonstructural protein 5) remain unknown.
Interferon-alpha (interferon) is a Food and Drug Administration-approved treatment for chronic HCV infection. The effects of interferon are mediated through different cellular inducible proteins, including double-stranded RNA-activated protein kinase (PKR) (Gale et al., 1997, Virology 230:217-227). Only 8 to 12% of patients with HCV genotype 1 have a sustained clinical virological response to interferon therapy (Carithers et al., 1997, Hepatology 26:83S-88S; Lindsay, 1997, Heptatology 26:71S-77S). Recently, combination therapy with interferon and the guanosine analogue, ribavirin, was shown to be superior to interferon monotherapy in producing sustained biochemical and virological responses (Poynard et al., 1998, Lancet 352:1426-1432). However, despite the significant improvement in rates of sustained response, as many as 60% of patients with high-titer HCV genotype 1 infection are nonresponsive to combination therapy. For example, the response rate in patients infected with HCV-1b is less than 40%. Similar low response rates for patients infected with prototype United States genotype, HCV-1a, have also been reported (Mahaney et al. 1994, Hepatology 20:1405-1411). In contrast, the response rate of patients infected with HCV genotype-2 is nearly 80% (Fried et al., 1995, Semin. Liver Dis. 15:82-91.) Expression of the entire HCV polyprotein has been shown to inhibit interferon-induced signaling in human U2-OS osteosarcoma cells (Heim et al., 1999, J. Virol. 73:8469-8475). It was not reported which HCV protein was responsible for this effect. The relationship between interferon-response and the nonstructural 5A (NS5A) sequence of HCV is controversial. Response to interferon therapy differs among the HCV subtypes, with the HCV-1b subtype being particularly resistant to interferon treatment (Alter et al., 1998, MMWR Recomm. Rep. 47 (RR-19):1-39). A comparison of the full length HCV nucleic acid sequence from interferon-resistant and interferon-sensitive viruses from HCV infected patients revealed missense substitutions corresponding to the carboxy terminus of NS5A (Enomoto et al., 1995, J. Clin. Invest. 96:224-230). The corresponding 40 amino acid region of NS5A (amino acids 2209-2248 of the HCV polyprotein) has been termed the interferon sensitivity determining region, or ISDR (Enomoto et al., 1995). The ISDR is enclosed within a region in the NS5A protein which can bind to and inhibit the function of PKR (Gale et al., Mol. Cell Biol., 1998, 18:5208-5218). Enomoto et al. (1996, N. Eng. J. Med. 334:77-81) proposed a model in which patients who respond to interferon-therapy are infected by viruses with multiple substitutions in the ISDR (compared to the interferon-resistant HCV 1b-J prototype sequence) whereas patients who fail interferon-therapy are infected by viruses with few substitutions in the ISDR.
Of the 25 studies that have published ISDR sequences from interferon-resistant and interferon-sensitive viruses, nine support the Enomoto model and conclude that, at the 5% significance level, the data provide sufficient evidence that interferon-response and substitutions in the ISDR are dependent (Enomoto et al., 1995, 1996; Chayama et al., 1997, Hepatology, 25:745-749; Kurosaki et al., 1997, Hepatology 25:750-753; Fukuda et al., 1998, J. Gastroenterol. Hepatol. 13:412-418; Saiz et al., 1998, J. Infect. Dis. 177:839-847; Murashima et al., 1999, Scand. J. Infect. Dis. 31:27-32; Sarrazin et al. 1999, J. Hepatol. 30:1004-1013; Sakuma et al., 1999, J. Infect. Dis. 180:1001-1009). The other 16 studies were unable to conclude that there is a correlation (Hofgartner et al., 1997, J. Med. Virol. 53:118-126; Khorsi et al., 1997, J. Hepatol. 27:72-77; Squadrito et al., 1997, Gastroenterology 113:567-572; Zeuzem et al., 1997, Hepatology 25:740-744; Duverlie et al., 1998, J. Gen. Virol. 79:1373-1381; Franguel et al., 1998, Hepatology 28:1674-1679; Odeberg et al., 1998, J. Med. Virol. 56:33-38; Pawlotsky et al., 1998, J. Virol. 72:2795-2805; Polyak et al., 1998, J. Virol. 72:4288-4296; Rispeter et al., 1998, J. Hepatol. 29:352-361; Chung et al., 1999, J. Med. Virol. 58:353-358; Sarrazin et al. 1999, J. Hepatol. 30:1004-1013; Squadrito et al., 1999, J. Hepatol. 30:1023-1027; Ibarrola et al., 1999, Am. J. Gastroenterol. 94:2487-2495; Mihm et al., 1999, J. Med. Virol. 58:227-234; Arase et al., 1999, Intern. Med. 38:461-466). Interestingly, seven of the nine studies that support a correlation are based on HCV isolates from Japan whereas 15 of the 16 studies that do not support a correlation are based on isolates from European and North American isolates. Although a statistically significant correlation between interferon response and ISDR sequence in North American and European studies are generally not found, there is evidence that a relationship does exist. When the intermediate and mutant classes of ISDR sequences from an individual study are combined, the response rates to interferon are higher than those in patients with the wild-type class of ISDR sequence (Herion and Hoofnagle, 1997, Hepatology 25:769-771).